The proposed studies are designed to determine the mechanism by which extracelluar enamel matrix proteins regulate initial enamel mineral formation and structural organization. Our working hypothesis is that through cooperative mechanisms involving protein self-assembly and the formation of a precursor mineral phase, higher order assemblies of amelogenin, in possible association with other matrix molecules, regulate the growth, shape, and organization of initial enamel mineral crystals. We propose that amelogenin super-assemblies guide the formation of linear arrays of amorphous calcium phosphate (ACP) nanoparticles that subsequently fuse and transform into ribbon-like apatitic crystals which serve as a template for the mature enamel structure. The long-term goal of this study is to provide fundamental insight into how matrix proteins control mineralization and structure in mineralized tissues, like enamel. Importantly, our findings should aid the development of bio-inspired materials and novel approaches for mineralized tissue repair and regeneration. Given the high prevalence of dental caries, there is a tremendous need for restorative procedures that are superior to those presently available. Hypotheses will be tested in vitro, primarily using native amelogenins that contain a single phosphorylated site that we have found to promote amelogenin[unreadable]s capacity of stabilize ACP for long periods of time. Four specific aims will be carried out using multiple approaches, including: dynamic light scattering, transmission electron microscopy, electron diffraction, Fouriertransform infrared spectroscopy, small angle x-ray scattering, small angle neutron scattering, and cryomicroscopy Specifically: Aim 1. In vitro studies will be carried out to test the hypothesis that full-length native amelogenin can regulate the formation of ordered bundles of hydroxyapatite (HA) crystals under appropriate kinetic conditions that promote the transformation of initially formed ACP nanoparticles to HA;Aim 2. In vitro studies will be carried out to test the hypothesis that the transformation of ACP stabilized by fulllength native amelogenin (P173) to ordered bundles of HA crystals can be induced in situ by proteolysis and/or by phosphatase;Aim 3. To determine the structure and mechanism of formation of higher-order assemblies of full-length native amelogenin, with particular emphasis on how this process is influenced by: 1) amelogenin phosphorylation;2) co-assembly with specific amelogenin degradation products;and 3) coassembly with specific non-amelogenins;and Aim 4. In vitro mineralization studies will be carried out using native and recombinant porcine amelogenins to test the hypothesis that higher-order assemblies of amelogenin control the formation of parallel arrays of mineral crystals through dynamic and cooperative interactions between protein assembly and mineralization. Longitudinal ultrastructural studies will be carried out using conventional TEM and cryo-TEM approaches.